EP0171072A2 - Méthode et appareil pour la détermination en phase solide d'une séquence de fragments d'acide nucléique - Google Patents

Méthode et appareil pour la détermination en phase solide d'une séquence de fragments d'acide nucléique Download PDF

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Publication number
EP0171072A2
EP0171072A2 EP85109906A EP85109906A EP0171072A2 EP 0171072 A2 EP0171072 A2 EP 0171072A2 EP 85109906 A EP85109906 A EP 85109906A EP 85109906 A EP85109906 A EP 85109906A EP 0171072 A2 EP0171072 A2 EP 0171072A2
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EP
European Patent Office
Prior art keywords
fragments
reaction
reaction vessels
sequencing
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP85109906A
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German (de)
English (en)
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EP0171072A3 (fr
Inventor
André Dr. Rosenthal
Hans-Dieter Dr. Hunger
Horst Kagelmaker
Monika Grätschus
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Akademie der Wissenschaften der DDR
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Akademie der Wissenschaften der DDR
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DD26599784A external-priority patent/DD228906B1/xx
Priority claimed from DD26599884A external-priority patent/DD228461B1/de
Priority claimed from DD27632885A external-priority patent/DD237329A1/de
Application filed by Akademie der Wissenschaften der DDR filed Critical Akademie der Wissenschaften der DDR
Publication of EP0171072A2 publication Critical patent/EP0171072A2/fr
Publication of EP0171072A3 publication Critical patent/EP0171072A3/fr
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • G01N2001/288—Filter punches
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • the invention relates to a method and a device for sequence analysis of nucleic acid fragments (DNA and RNA) in molecular biology and genetic engineering or for sequence analysis of oligodeoxy and oligoribonucleotides after chemical synthesis.
  • a solid phase method for the sequencing of DNA fragments has already been specified (SA Chuvpilo and VV Kravchenko, Bioorg. Khim 9 (12) (1983) 1634 - 1637), which, however, is only suitable for sequencing long DNA sections and in which a commercial available DEAE paper Whatman DE 81 is used.
  • This method comprises the following steps: 1) immobilization of the 5'- or 3'-labeled DNA fragment on the support; 2) washing; 3) chemical modification reactions without excess of reagent (i.e. not in a reaction vessel) according to Maxam and Gilbert (A.M. Maxam and W. Gilbert, Methods in Enzymol.
  • the described method cannot be automated due to the disadvantages described above, on the other hand, even when carried out manually, large numbers cannot be sequenced at the same time. Short nucleic acid fragments (DNA and RNA oligomers) cannot be sequenced at all.
  • the invention is based on the object of binding long and short nucleic acid fragments to a solid phase in such a way that fully automated or safe manual handling of the immobilized products is possible and the nucleic acid fragments can be determined quickly, without confusion and in large numbers simultaneously.
  • the object is achieved according to the invention with a solid phase sequencing method for nucleic acid fragments using a novel surface carrier with anion exchange properties and with a new device for carrying out the method.
  • the immobilization of the or the marked fragments on the surface carrier is carried out in a manner known per se, e.g. B. by dripping or by electroelution from gels (4 carriers per fragment). These supports are washed and then the immobilized fragment or several immobilized fragments are chemically modified simultaneously. The chemical modification reactions take place with or without excess reagent. Without excess of reagent, the surface carriers are on a chemically inert matrix, on which just enough reagents with micropipettes are applied to a surface carrier that it is only moistened.
  • the sorted fragments are reacted individually with piperidine and eluted with chemical, ionic or electromagnetic means.
  • the compounds of the formula I can advantageously be intermediately reacted by compounds of the general formula II with R and Y as above with tertiary amines of the general formula III generated and used accordingly.
  • shaped macromolecular masses with chemically active fillers such as. B. mechanically stable cellulose papers, nonwovens and fabrics, implemented and in this way mechanically stable Flat supports with anion exchange properties and with a high binding capacity for nucleic acids of up to 120 ⁇ g / cm can be obtained.
  • anion exchangers which can be eluted photolytically, ie using electromagnetic radiation, which means an advantage with regard to the automatability of the method.
  • All of the above-mentioned anion exchangers are additionally ionic agents, ie with solutions of high ionic strength of salts, such as. B. NH 4 HCO 3 , (NH 4 ) 2 CO 3 , Et 3 NHC0 3 , Et 3 NHAc, NH 4 Ac, K 2 HPO 4 , NaCl and others.
  • the anion exchangers described have a high binding capacity and, particularly in terms of their surface shape, they have excellent mechanical stability. For this reason, the use of the shaped surface carriers with anion exchange properties is particularly indicated in further molecular biological and biochemical research.
  • the surface carriers according to the invention can be eluted not only by ionic elution but also by chemical means or by electromagnetic radiation.
  • 1 or n 5'- or 3'-labeled long or short nucleic acid fragments are applied to 4 or 4 xn individual mechanically stable surface supports (approx. 1 to 2 x 1 to 2 mm) with anion exchange properties.
  • n 5'- or 3'-labeled long or short nucleic acid fragments are each immobilized on 4 individual, but geometrically larger, surface carriers, so that all n fragments are each fixed to a surface carrier with a safety margin.
  • All surface supports must be specially marked. Subsequent washing of the surface supports is carried out twice in succession in water and in ethanol, removing salts or other contaminants. Then they are briefly dried in air or, if necessary, with heated air.
  • the chemically modified surface supports are carefully washed twice in succession with water and ethanol. This is done manually by immersing the surface supports with the tweezers in the appropriate solvents, placing them between the blotting paper and drying them with pressure. The surface supports resulting in a large number according to c) can be washed together, but must then be sorted out again.
  • the Carrier in the reactors with frit also washed by continuous or discontinuous metering of water and ethanol. All surface supports with only one immobilized fragment are then treated individually with a fresh 10% strength aqueous piperidine solution at 90 ° C. for 15 to 45 minutes. The DNA strand breaks and the carrier is eluted at the same time.
  • the geometrically larger surface supports with n immobilized fragments are cut in such a way that n individual pieces result from a larger support. These are treated individually with piperidine as above,.
  • the device according to the invention is described below, with the aid of which a simultaneous immobilization of a large number of nucleic acid fragments on a surface support, a simultaneous chemical further treatment, simultaneous punching out of the solid-phase immobilizates, a further simultaneous chemical treatment and simultaneous aftertreatments such as washing, high and low temperature treatment and centrifuging possible are.
  • the device has sequencing blocks with associated covers, surface support holders, sample dispensers and punches. Both the lid and the surface holder as well as the sample dispenser and punch can only be combined with the sequencing block in one position by attaching markings and guides, so that the samples cannot be mixed up.
  • the sequencing block consists of a cuboid or cube-shaped basic block, although other shapes are also possible, embedded or incorporated in the reaction vessels. The number of reaction vessels depends according to the scope of the individual samples to be analyzed.
  • a marking indicates the constellation of the assignment of the other parts of the device that are required according to the invention due to the process sequence.
  • the lid is designed so that it fits exactly on the sequencing block and tightly closes each inserted reaction vessel. It consists of a non-deformable rigid cover, to which an elastic seal is attached, which presses on the reaction vessels, and fastening elements for fixing to the sequencing block.
  • the device according to the invention includes a sample dispenser for the simultaneous application of the liquid samples to the surface carrier.
  • the sample dispenser has capillaries of the same length, which are inserted in a capillary holder and have the same distribution pattern as the reaction vessels, each capillary therefore being assigned to a different reaction vessel.
  • markings and guides ensure that the capillaries are always assigned to the same sample vessel.
  • the punch used according to the invention with the aid of which the sample immobilizates located on the surface carrier are punched out and simultaneously transferred into the reaction vessels of the sequencing block, consists of a plate with inserted punching pins which have the same distribution pattern as the reaction vessels in the sequencing block.
  • the sample dosing and the punching takes place after the surface carrier has been inserted into a surface carrier holder which engages in the prescribed, unique position on the sequencing block.
  • the sample can also be dosed outside the sequencing block.
  • a basic equipment of the device expediently includes five sequencing blocks, four lids, four flat support holders, a sample dispenser and a punch.
  • all essential operations take place with or in the sequencing block. This is a remarkable advantage that enables economic work and eliminates confusion.
  • Cellulose paper for example Whatman 540 paper, is activated with cyanuric chloride in accordance with EP 134 025 and packaged immediately.
  • the paper (5 x 7 cm) that has been surface-treated in this way is removed for use from its special packaging (welded foil, which is stored under N 2 gassing at -20 ° C) and placed in a glass reaction chamber in which 2 mmol ( 450 mg) of triethyl-1-aminoethyl-ammonium bromide in 7 ml of acetonitrile.
  • the reaction chamber is then shaken at room temperature for about 12 hours.
  • the paper is washed several times with water and dried between blotting paper.
  • aqueous buffer systems can also be used.
  • Cellulose paper (Whatman 540 paper) is surface-activated as in Example 1 with cyanuric chloride and then placed in a glass reaction chamber in which 2 mmol (410 mg) of 1-bromo-2-aminoethane hydrobromide in 7 ml of acetonitrile were placed. The reaction is then started with the addition of 5 ml of triethylamine. The reaction chamber is sealed with parafilm and shaken at room temperature for about 12 hours. After the reaction has ended, the paper is washed and dried as in Example 1.
  • Example 2 400 mg of crystalline cellulose (Whatman cellulose) surface-activated according to Example 2 is reacted with 2 mmol (574 mg) of p-aminobenzyl-triethylammonium bromide in 7 ml of acetonitrile, as described in Example 2, and worked up.
  • Example 1 Surface-activated cellulose paper (Whatman 540 paper, 5 x 7 cm) from Example 1 is mixed with 2 mmol (540 mg) of p-aminobenzyl bromide hydrobromide in 7 ml of acetonitrile in a reaction vessel, whereupon the reaction is started with 5 ml of triethylamine. After 12 h of reaction at room temperature, the paper is washed and dried as in Example 1.
  • Example 4 400 mg of crystalline cellulose (Whatman cellulose) which has been surface-activated according to Example 2 is reacted analogously to Example 4 with 2 mmol (540 mg) of p-aminobenzyl bromide hydrobromide in acetonitrile and with triethylamine and, after the reaction has ended, washed and dried.
  • Example 2 Surface-activated cellulose paper (Whatman 540 paper, 5 x 7 mm) from Example 1 is mixed with 2 mmol glycine (o-nitro-p-methyl-triethylammonium) bromide) -benzyl ester in 7 ml of acetonitrile reacted for 12 h at room temperature in a reaction chamber and worked up further as in Example 1.
  • glycine o-nitro-p-methyl-triethylammonium bromide
  • oligonucleotide 10 - 50 pmol oligonucleotide (or longer fragment) are labeled with ⁇ 32 P-ATP and polynucleotide kinase at the 5 'end according to conventional methods.
  • the total volume is applied to, for example, a 20-part polyacrylamide gel, which may contain urea (7 M), and gel electrophoresis of ATP and other impurities such as
  • the desired DNA band is made visible by short autoradiography on X-ray film and cut out of the gel.
  • the gel piece is crushed and transferred to an Eppendorf cone.
  • the labeled oligonucleotide is recovered by eluting twice with water at a temperature between 37 and 60 ° C for 30 minutes.
  • 1-2 g of the aqueous solution obtained under 1. which optionally contains salts and urea in addition to the labeled oligonucleotide, are dripped onto at least four pieces of the surface support of approximately 2 ⁇ 2 mm (Examples 1-10). After briefly drying the pieces of paper at room temperature or with warmed air, the dropping is repeated until approx. 10,000 to 50,000 cpm per surface area are measured on a scintillation measuring device. The papers are then washed with tweezers twice in succession in water and ethanol (approx. 1 min) and dried briefly under pressure after each washing operation between blotting paper.
  • reaction times are 10 minutes for the G reaction and 20 minutes for all other reactions.
  • the T> C reaction with osmium tetroxide 80 ⁇ l of a 5 mM Os0 4 solution + 1 ⁇ l pyridine, 15 min at 0 ° C
  • the A + G reaction with piperidine formate 80 ⁇ l piperidine formate pH 2.1 h at 37 ° C
  • diethyl pyrocarbonate 150 ⁇ l
  • a buffer 50 mM sodium acetate pH 5 + 1 mM EDTA
  • 5 to 10 ⁇ l of a freshly prepared 10% DEPC solution in ethanol 20 min at 90 ° C
  • the T + C and C reactions with hydrazine according to Maxam and Gilbert or the A> C reaction with 1.2 M NaOH solution lead to complete loss of radioactivity and can therefore not be used will.
  • the following losses occur during the above-mentioned modification reactions: 20% in the G-, 50% in the A + G-, 0% in the T> C- and 50-80% in the C-reaction. These are compensated for by double or quadruple radioactivity in the A + G or C reaction.
  • the modification reactions are ended by removing the papers from the reaction vessels with tweezers and washing them twice in succession with water and ethanol.
  • the 20 paper carriers are individually placed in 20 new Eppendorf hats. After adding 50 ⁇ l of a 10% strength aqueous piperidine solution, they are kept at 90 ° C. for 30 minutes. After the reaction has ended, the papers are removed from the Eppendorf cones with tweezers, and the solutions are kept at -200 ° C for 1 min. B. in liquid air, stored and lyophilized in vacuo (about 1 h). The lyophilization step is repeated twice with 10 to 20 ⁇ l of water (in each case approx. 30 min). The samples are then ready for gel electrophoresis application.
  • Example 11 Marking and immobilization of five Pentadeca nucleotides d (TTCTTCTACACACCC), d (TGATCAGATGGCTTT), d (CTCCTGGCCATTCCT), d (GGGTACCCAGAAGTC) and d (TCGCTGAGATCACCA) are carried out as described in Example 11 (1st and 2nd).
  • the chemical modification reactions are now only carried out in four Eppendorf cones, each containing five paper carriers.
  • the reaction conditions and the subsequent washing operations are as in Example 11 (3.).
  • the previously labeled supports are sorted out again, so that four individual papers per oligonucleotide are again present and can be brought individually into an Eppendorf cone for the subsequent piperidine reaction.
  • the piperidine reaction was carried out analogously to Example 11 (4.).
  • the four larger surface supports are subjected to the modification reactions already described in larger reaction vessels (corresponding to the size of the surface supports).
  • the four (2 x 20 mm) flat supports are cut with scissors into five smaller (2 x 2 mm) pieces of paper and placed individually in a total of 20 Eppendorf cones for the piperidine reaction.
  • the piperidine reaction and the lyophilization are carried out as described above.
  • a DNA / RNA sequencing according to the invention automat is constructed as follows: In the simplest case for sequencing only one nucleic acid fragment, four thermostattable reactors equipped with a frit and with a maximum capacity of 250 ⁇ l are arranged in parallel with meterable inlet and outlet. The four individual surface supports (2 x 2 mm) with the immobilized labeled nucleic acid fragments are introduced into the reactors. Now the operations are carried out automatically according to a predetermined program: 1) washing; 2) chemical modification reactions; 3) washing; 4) Piperidine reaction (elution). For this purpose, the individual reagents according to Example 11 (3.) are added via metering pumps and remain in the reactor for a specified time with the drain valve closed.
  • the valves of the reactors are opened and the reagents drain off, if appropriate, under a slight air pressure. All washing steps with water and ethanol can be carried out continuously (with the drain valve open, the above solvents are alternately fed into the reactors) or discontinuously (100 V l of the above solvents are fed into the reactor with the drain valve closed and remain there for a short time before they are used again be drained). During this continuous washing or at the end of the continuous washing process, the supports can optionally be dried with heated air for a few seconds.
  • the total time of the operation cycle is approximately 2.5 hours. In the second case with separate lyophilization, the total time is approximately 20 to 30 minutes.
  • the samples are then ready for gel electrophoresis.
  • the four reactors must have different dimensions.
  • the base area of a reactor is approx. 0.5 x 6 cm for 8 to 15 fragments to be sequenced.
  • the four (0.4 x 5.5 cm) large surface supports with applied 8 to 15 fragments are again introduced into the reactors, after which steps 1) and 2) are carried out, of course with larger volumes of reagents. After draining the reagents of the modification reactions, a wash follows (Operation 3).
  • the dried surface carriers are then automatically cut into the individual, smaller pieces in a predetermined manner and at the same time are introduced into different piperidine reactors with frit and drain valve (approx. 50 reactors).
  • the best way to do this is with four movable modification reactors, which are after step 3) lower by 180 ° and then open on the reactor cover.
  • the piperidine reactions proceed analogously, as already described. After the reaction has ended, the piperidine solutions with the eluted fragments are transferred to the Eppendorf cones (approx. 50 pieces) connected to the reactors by means of PTFE tubing (about 50 pieces) and then lyophilized.
  • the fragments can also be eluted with 1 to 2 M solutions of NH 4 HC0 3 , ( NH 4 ) 2 CO 3 , NH 4 Ac, Et 3 NHAc and others before the piperidine reaction.
  • the elution is achieved in that the surface supports are mixed twice in 50 ml of the above-mentioned salt solutions at 60 ° C. in reaction vessels such as Eppendorf cones.
  • the eluates are lyophilized, and the salts can be volatilized with aqueous ethanol upon repeated lyophilization.
  • the salts can also be removed by ethanol precipitation.
  • the usual piperidine reaction then takes place in a homogeneous phase.
  • the basic equipment of the device for carrying out the solid phase sequencing according to the invention consists of five sequencing blocks 1, four lids 2, a sample dispenser 3, a punch 5 and four surface mount holders 4 (cf. FIGS. 1-3).
  • the sequencing block 1 has a cuboid basic block 1.1, in which the reaction vessels 1.2 and a locking device 1.3 are embedded.
  • the lid 2 fits exactly on the sequencing block. It has an elastic cover 2.1 fastened on the rigid cover 2.2, which has stopper-like elevations 2.4 corresponding to the number of reaction vessels 1.2.
  • the fasteners 2.3 are arranged at the corners. These are usually two barriers that are rotated into the threaded holes provided in the sequencing block.
  • the base support 4 includes a base plate 4.1 with the recess 4.2, a guide plate 4.3 and the locking 4.5.
  • the guide plate and base plate Corresponding to the number and the distribution pattern of the reaction vessels 1.2, the guide plate and base plate have bores 4.4 which allow the insertion of the punch pins 5.2 and the capillaries 3.2.
  • the diameter of the holes 4.4 is smaller than the inside diameter of the reaction vessels 1.2.
  • the sample dispenser consists of the capillary holder 3.1 and the capillaries 3.2.
  • the capillary holder 3.1 a rectangular plate corresponding to the area of the sequencing block 1, has as many holes and in the same arrangement as the reaction vessels in the sequencing block act or are arranged.
  • the capillaries 3.2 are slidably inserted in the bores, the orifices 3.3 of the capillaries 3.2 lying on one plane and the distance between the orifices and the capillary holder being at least as large as the reaction vessels 1.2 are deep.
  • the diameter of the capillaries 3.2 is slightly smaller than the diameter of the bores 4.4 of the surface carrier holder 4.
  • the punch 5 has the same rectangular shape as the sequencing block.
  • the stamping plate 5.1 consists of the stamping plate 5.1 and the punching pins 5.2 embedded therein, the number and distribution of which correspond to the number and distribution of the bores 4.4 in the surface carrier holder.
  • the diameter of the punch pins is chosen so that they fit exactly into the holes 4.4.
  • the length of the punch pins 5.2 is at least as large as the base plate 4.1 and guide plate 4.3 are thick.
  • the samples to be examined are filled into the reaction vessels 1.2 of a sequencing block 1.
  • an approximately uniform filling of the capillaries is achieved by immersing the capillaries in the aqueous solution due to the acting capillary forces (FIG. 2a).
  • the sample dispenser is then pressed onto the absorbent surface support 4.6, which is immovably in the recess 4.2 of the base plate 4.1.
  • sample liquid is transferred to the carrier.
  • the surface carrier receives a sample pattern that corresponds to the arrangement pattern of the reaction vessels. The distance between the applied samples or between the reaction vessels is so chosen that running into each other is impossible (Fig. 1a).
  • the piperidine solution prescribed by the process is added to the reaction vessels of all sequencing blocks, whereupon the lids 2 are placed and all blocks are subjected to a temperature treatment of 90 ° C. at the same time.
  • the DNA strand breaks and the carrier is eluted at the same time.
  • the further process steps such as shaking, separating the carrier fragments, centrifuging, cooling and lyophilizing, take place in or with the sequencing blocks (cf. FIGS. 4: 13-19).
  • the samples are further processed for separation and visualization by gel electrophoresis. A mix-up of samples is impossible, because by placing markings only an assignment of the elements of the device such as cover 2, sample dispenser 3, surfaces carrier holder 4 and punch 5 to the sequencing block is possible.
  • Another great advantage is the simultaneous treatment of the surface support fragments 20 or the eluted samples until the time of application to the gel electrophoresis without removal from the sequencing block. This results in a considerable reduction in the analysis time and a high sample throughput is achieved by selecting the number of reaction vessels in the sequencing block.

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  • General Physics & Mathematics (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
EP85109906A 1984-08-06 1985-08-06 Méthode et appareil pour la détermination en phase solide d'une séquence de fragments d'acide nucléique Ceased EP0171072A3 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
DD265998 1984-08-06
DD265997 1984-08-06
DD26599784A DD228906B1 (de) 1984-08-06 1984-08-06 Verfahren zur festphasen-sequenzierung von nucleinsaeurefragmenten
DD26599884A DD228461B1 (de) 1984-08-06 1984-08-06 Verfahren zur herstellung von anionenaustauschern
DD276328 1985-05-14
DD27632885A DD237329A1 (de) 1985-05-14 1985-05-14 Einrichtung zur festphasensequenzierung von nucleinsaeurefragmenten

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Publication Number Publication Date
EP0171072A2 true EP0171072A2 (fr) 1986-02-12
EP0171072A3 EP0171072A3 (fr) 1988-09-07

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EP85109906A Ceased EP0171072A3 (fr) 1984-08-06 1985-08-06 Méthode et appareil pour la détermination en phase solide d'une séquence de fragments d'acide nucléique

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US (2) US4849077A (fr)
EP (1) EP0171072A3 (fr)
HU (1) HU198796B (fr)
YU (1) YU125585A (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0206411A2 (fr) * 1985-06-17 1986-12-30 The Board Of Trustees Of The University Of Illinois Chromatographie en phase renversée pour la détermination d'une séquence d'oligonucléotides
EP0244207A1 (fr) * 1986-04-30 1987-11-04 Toray Industries, Inc. Méthode de détection et appareil
EP0269764A1 (fr) * 1986-12-01 1988-06-08 Molecular Biosystems, Inc. Procédé pour augmenter la sensibilité des essais d'hybridation des acides nucléiques
EP0303459A2 (fr) * 1987-08-11 1989-02-15 The President And Fellows Of Harvard College Séquenage multiplexe
WO2008003693A1 (fr) * 2006-07-04 2008-01-10 Tecan Trading Ag Dispositif de collecte pour des échantillons pertinents d'un point de vue biologique
EP2855020A2 (fr) * 2012-06-01 2015-04-08 Vycap B.V. Dispositif de diagnostic à micro-tamis pour l'isolement et l'analyse de cellules individuelles

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Publication number Priority date Publication date Assignee Title
US5102785A (en) * 1987-09-28 1992-04-07 E. I. Du Pont De Nemours And Company Method of gene mapping
JPH02157655A (ja) * 1988-12-09 1990-06-18 Seiko Instr Inc 反応容器
US5081584A (en) * 1989-03-13 1992-01-14 United States Of America Computer-assisted design of anti-peptides based on the amino acid sequence of a target peptide
US7049102B1 (en) 1989-09-22 2006-05-23 Board Of Trustees Of Leland Stanford University Multi-gene expression profile
US5545522A (en) 1989-09-22 1996-08-13 Van Gelder; Russell N. Process for amplifying a target polynucleotide sequence using a single primer-promoter complex
AU2674092A (en) * 1991-09-09 1993-04-05 Baylor College Of Medicine Method and device for rapid dna or rna sequencing determination by a base addition sequencing scheme
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US4882127A (en) 1989-11-21
US4849077A (en) 1989-07-18
HUT39019A (en) 1986-07-28
YU125585A (en) 1988-06-30

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